Cancellous bone product including viable osteogenic cells

ABSTRACT

A bone implant comprising cancellous bone that is essentially free of blood cells, and which has been treated with at least one loosening agent, such as collagenase or a digestive enzyme, for a time and at a concentration to loosen the osteogenic cells in the cancellous bone matrix. The osteogenic cells in the matrix are viable cells. The treatment of the cancellous bone with at least one loosening agent enables the osteogenic cells to be more available for carrying out their osteogenic function and to provide for an increased rate of bone formation. Such implant also may include demineralized bone, such as demineralized cortical bone, which enhances the bone regenerative capacity of the cancellous bone.

This application is which is a continuation of U.S. patent applicationSer. No. 15/622,985 filed Jun. 14, 2017, which is a continuation ofpatent application Ser. No. 12/150,513 filed Apr. 28, 2008, which was acontinuation-in part of application Ser. No. 11/799,606, filed May 2,2007 which claimed priority to provisional application Ser. No.60/831,723, filed Jul. 18, 2006, and Ser. No. 60/798,474, filed May 8,2006, the contents of which are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

This invention relates to bone implants comprising cancellous boneuseful in the treatment or prevention of bone diseases, disorders,defects, or injuries.

BRIEF SUMMARY OF THE INVENTION

This invention relates to bone implants which include cancellous bone.More particularly, this invention relates to bone implants that includecancellous bone that has been treated with at least one loosening agentin order to loosen osteogenic cells in the bone matrix. The cancellousbone also is essentially free of blood cells.

This invention also relates to bone implants which include thecancellous bone hereinabove described, and demineralized bone. Moreparticularly, this invention also relates to bone implants which includethe cancellous bone hereinabove described, and bone, such as, forexample, cortical bone, which has been treated with at least onedemineralization agent for a time and at a concentration to exposeosteoinductive proteins in the bone matrix.

Bone implants which include cancellous bone have been used in a varietyof procedures and treatments, including bone fusions such as spinefusions, disc augmentations in the spine, and bone fill applicationsemployed in the treatments of diseases, disorders, or injuriesincluding, but not limited to, avascular osteonecrosis, osteosarcoma,acute fractures and fracture non-unions, as well as for boneregeneration for orthopedic implants. Such bone in general may beharvested from any source of cancellous bone, including vertebrae, theiliac crest, femur, tibia, or ribs.

Cancellous bone implants in general have included, in addition toosteocytes and osteogenic cells, blood cells including hematopoieticcells. In some implants, the implants have been treated in order topreserve the viability of all the cells in the implant, while in otherimplants, the viability of the bone cells, including the osteogeniccells, has been destroyed.

The present invention provides a bone implant which includes cancellousbone including viable osteogenic cells which are made more available forcarrying out their osteogenic function.

In accordance with an aspect of the present invention, there is provideda bone implant comprising cancellous bone. The cancellous bone isessentially free of blood cells, and has been treated with at least oneloosening agent for a time and at a concentration to loosen theosteogenic cells in the cancellous bone matrix. The osteogenic cells inthe bone matrix are viable cells.

The term “loosening agent” as used herein, means an agent which may becontacted with a bone matrix for a time and at a concentrationsufficient to loosen osteogenic cells in the cancellous bone matrixwithout releasing the osteogenic cells from the cancellous bone matrix.An exemplary loosening agent useful in accordance with the presentinvention comprises collagenase. Another exemplary loosening agentuseful in accordance with the present invention comprises a digestiveenzyme. In some embodiments, the digestive enzyme is selected from thegroup consisting of trypsin, amylase, lipase, and combinations thereof.In some embodiments, the digestive enzyme is trypsin. In someembodiments, the digestive enzyme is amylase. In some embodiments, thedigestive enzyme is lipase. Another exemplary loosening agent useful inaccordance with the present invention comprises a combination ofcollagenase and a digestive enzyme. For a time and at a concentration toloosen the osteogenic cells contained in the bone matrix, but notrelease the osteogenic cells from the bone matrix.

The term “osteogenic cell”, as used herein, means any type of cellhaving osteoprogenitor potential, that is, any type of cell that iscapable of differentiating into a bone cell.

The term “D90”, as used herein, refers to the size of the 90thpercentile (by number) particle in a cumulative size distribution of allparticles sampled.

Although the scope of the present invention is not to be limited to anytheoretical reasoning, when the bone implant is essentially free ofblood cells and has been treated with a loosening agent in order toloosen the osteogenic cells in the cancellous bone matrix, suchosteogenic cells become more available for or are more disposed towardcarrying out their osteogenic function, and provide for an increasedrate of bone formation. Thus, such bone implants are capable ofgenerating or “growing” bone directly and provide for improved implantsvis-a-vis prior art implants, including previously produced cancellousbone implants, and ceramic implants.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention are useful in the treatment orprevention of bone diseases, disorders, defects, or injuries.

In one embodiment, the present invention comprises a bone implant thatcomprises: cancellous bone matrix, said cancellous bone matrix beingessentially free of blood cells, said cancellous bone matrix having beentreated with at least one loosening agent for a time and at aconcentration to loosen osteogenic cells in the cancellous bone matrix,said osteogenic cells in the cancellous bone matrix being viable cells.

In one embodiment, the present invention comprises a bone implant thatcomprises: (i) cancellous bone matrix, said cancellous bone includingcancellous bone which is essentially free of blood cells and having beentreated with at least one loosening agent for a time and at aconcentration to loosen osteogenic cells in the cancellous bone matrix,said osteogenic cells in the cancellous bone matrix being viable cells;and (ii) cortical bone matrix which has been treated with at least onedemineralization agent for a time and at a concentration to exposeosteoinductive proteins present in the cortical bone matrix.

In some embodiments, the at least one demineralization agent ishydrochloric acid.

The cancellous bone is treated with the loosening agent to loosen theosteogenic cells in the cancellous bone matrix. In one embodiment, thecancellous bone is treated with at least one loosening agent at aconcentration of from about 0.1 mg/ml to about 3.0 mg/ml, and in anotherembodiment from about 1.0 mg/ml to about 3.0 mg/ml. The cancellous boneis treated with at least one loosening agent for a period of time toloosen the osteogenic cells in the cancellous bone matrix, but notrelease the osteogenic cells from the cancellous bone matrix. In oneembodiment, the cancellous bone is treated with at least one looseningagent for about 5 min. to about 3 hrs. In another embodiment, thecancellous bone is treated with at least one loosening agent for about 5min, to about 30 min. In yet another embodiment, the cancellous bone istreated with at least one loosening agent at a concentration of about 1mg/ml for about 10 minutes.

Although Applicants do not intend to be limited to any theoreticalreasoning, it is believed that, by treating the bone with at least oneloosening agent as hereinabove described, such treatment provides for apartial, but not complete, digestion of the components of the bonematrix (such as collagen, for example). Such partial digestion of thecomponents of the bone matrix, loosens, but does not release, osteogeniccells from the matrix, thereby making such cells, as noted hereinabove,more available for or more disposed toward carrying out their osteogenicfunction.

In general, the cancellous bone is harvested from any cancellous bonebearing source. Such sources include, but are not limited to, vertebralbodies in the spine, the iliac crest, femur, tibia, and ribs. Thecortical shell of the bone is removed, and then the bone is cut ormilled into desired pieces or shapes. For example, the bone may be cutor milled into bone chips, or may be cut into wedges or plugs, or may beformed into pellets.

The bone then is washed to remove blood cells, such as red blood cellsand hematopoietic cells. After the bone is washed, the bone is treatedwith at least one loosening agent, such as collagenase or a digestiveenzyme. As noted hereinabove, the bone is treated with a loosening agentfor a time and at a concentration to loosen the osteogenic cellscontained in the bone matrix, but not release the osteogenic cells fromthe bone matrix. In one embodiment, the bone is treated with theloosening agent at a concentration of about 0.1 mg/ml to about 3 mg/ml,and in another embodiment from about 1.0 mg/ml to about 3.0 mg/ml. Inanother embodiment, the bone is treated for about 5 min, to about 3 hrs.In another embodiment, the bone is treated for about 5 min. to about 30min. In yet another embodiment, the bone is treated with the looseningagent at a concentration of about 1.0 mg/ml for about 10 minutes.

In one embodiment, subsequent to the treatment with the loosening agentthe bone is treated with one or more antibiotics or one or moreantimycotics in order to reduce the level of bioburden within the bone.Antibiotics which may be employed include, but are not limited to,gentamicin; vancomycin; penicillins; macrolide antibiotics, such aserythromycin; sulfa-based antibiotics, and combinations thereof.Antimycotics which may be employed include, but are not limited to,amphotericin, fluconazole, and combinations thereof.

After antibiotic or antimycotic treatment, when chipped or milled boneis employed, the chipped or milled bone, if desired, may be filteredthrough sieves in order to retain pieces of the bone which have adesired size.

After the bone has been washed, treated with the loosening agent andtreated with an antibiotic or antimycotic, the bone then may be added toan appropriate preservation medium, such as a cryopreservation orvitrification medium, in which the bone may be preserved and stored, andthe osteogenic cells contained therein will remain viable. In oneembodiment, the preservation medium may include one or more of glycerol,dimethylsulfoxide, and DMSO, In one embodiment, the preservation mediumenables the treated bone to be frozen at temperatures as low as about−140° C. and as high as about −20° C. while maintaining the viability ofthe osteogenic cells. The present invention also contemplates that thecancellous bone treated with a loosening agent may be combined with bonethat has not been treated with a loosening agent prior to packaging as afinal product. For example, the cancellous bone treated with theloosening agent may be mixed with bone that has not been treated with aloosening agent, such as allograft bone chips, fragments or powder, ornucleus pulposus. For example, in one embodiment, the bone treated withthe loosening agent is admixed with demineralized bone prior toformulation into a final product. In another embodiment, the finalproduct includes about 50 vol. % treated bone, and about 50 vol. %untreated bone.

The bone implant treated with the loosening agent may be administered toan animal in an amount effective to treat a bone disease, disorder,defect, or injury in the animal. The animal may be a mammal, includinghuman and non-human primates. In one embodiment, the animal is a human.

Bone diseases, disorders, defects, or injuries which may be treated bythe bone implant treated with the loosening agent include, but are notlimited to, degenerative disc disease, avascular osteonecrosis,osteosarcoma fractures, and fracture non-unions. The bone implanttreated with the loosening agent also may be employed in bone fusions,such as spine fusions, as well as in disc augmentation, and for boneregeneration in orthopedic implants.

The bone implant treated with the loosening agent may be administereddirectly to the site of the bone disease, disorder, defect, or injury.Depending upon the form and shape of the implant, the implant may beinjected directly into the site affected by the bone disease, disorder,defect, or injury, or the implant may be packed directly into the siteaffected by the bone disease, disorder, defect, or injury. The implanthas a sufficient consistency' such that the implant will be retained atthe implantation site long enough for initial bone formation,osteoinductive signaling, and host cell attachment to occur. The presentinvention also contemplates that the bone implant may be employed inconjunction with devices employed in the treatment of bone diseases,defects, disorders, and injuries, such as, for example, orthopedic cagedevices, ceramics, or plates which may be employed in the spine or inbones to promote bone growth and fusion. Furthermore, the bone implantmay be used in conjunction with an autologous bone graft. The boneimplant also may be administered with antibiotics, such as thosehereinabove described, antimycotics, or anti-inflammatory agents. Inanother embodiment, the bone implant may be administered in combinationwith osteoinductive factors such as, for example, bone morphogenicproteins, or BMPs, such as BMP-2 and BMP-7, and platelet-derived growthfactor (PDGF), which enhance the osteogenic potential of the boneimplant. It is to be understood, however, that the scope of the presentinvention is not intended to be limited to the treatment of anyparticular bone disease, defect, disorder, or injury, or to anyparticular form or any particular method of administration of the boneimplant.

As noted hereinabove, the cancellous bone may be admixed withdemineralized bone. Thus, in accordance with another aspect of thepresent invention, there is provided a bone implant comprising (i)cancellous bone, said cancellous bone including cancellous bone which isessentially free of blood cells, as hereinabove described, which hasbeen treated with at least one loosening agent for a time and at aconcentration to loosen the osteogenic cells in the cancellous bonematrix, wherein the osteogenic cells in the bone matrix are viablecells; and (ii) bone which has been treated with at least onedemineralization agent for a time and at a concentration to exposeosteoinductive proteins present in the bone matrix.

In another embodiment, the cancellous bone, in addition to the treatedcancellous bone hereinabove described, further comprises bone that hasnot been treated with a loosening agent.

Although the scope of the present invention is not to be limited to anytheoretical reasoning, Applicants have discovered that the demineralizedbone enhances the bone regenerative capacity of the cancellous bone.

In one embodiment, the bone which is treated with the at least onedemineralization agent is cortical bone.

In one embodiment, the at least one demineralization agent which is usedto treat the bone, such as, for example, cortical bone, includes, but isnot limited to, any acidic solution. In another embodiment, the at leastone demineralization agent is hydrochloric acid.

The bone, such as, for example, cortical bone, is treated with the atleast one demineralization agent for a time and at a concentration toexpose osteoinductive proteins present in the bone matrix. Suchosteoinductive proteins include, but are not limited to, a heterogeneousmixture of bone morphogenic proteins and growth factors. In oneembodiment, the bone, such as, for example, cortical bone, is treatedwith the at least one demineralization agent, such as hydrochloric acid,at a concentration of from about 0.1N to about 12N. In anotherembodiment, the bone, such as, for example, cortical bone, is treatedwith the at least one demineralization agent, such as hydrochloric acidat a concentration, for example, of about 0.5N.

In one embodiment, the bone, such as, for example, cortical bone, istreated with the at least one demineralization agent for a period oftime of from about 1 minute to about 72 hours. In another embodiment,the bone, such as, for example, cortical bone, is treated with the atleast one demineralization agent for a period of time of from about 15minutes to 180 minutes. In another embodiment, the bone, such as, forexample, cortical bone, is treated with the at least onedemineralization apart for a period from about 30 minutes to about 120minutes. In another embodiment, the bone, such as, for example, corticalbone, is treated with the at least one demineralization agent for aperiod of time of from about 50 minutes to about 70 minutes.

In one embodiment, the bone, such as, for example, cortical bone,comprises particles which have a D₉₀ of less than about 1,500 microns.In another embodiment, the particles have a D₉₀ of about 125 microns toabout 1,500 microns. In yet another embodiment, the particles have a D₉₀of about 780 microns to about 1,500 microns. In another embodiment, theparticles have a D₉₀ of about 125 microns to about 780 microns. Inanother embodiment, the particles have a D₉₀ of about 600 microns toabout 900 microns. In another embodiment, the particles have a D₉₀ ofabout 700 microns to about 800 microns. In another embodiment, theparticles have a D₉₀ of about 780 microns.

In still another embodiment, the bone, such as, for example, corticalbone, comprises a first portion comprising particles having a D₉₀ fromabout 780 microns to about 1,500 microns, and a second portioncomprising particles having a D₉₀ from about 125 microns to about 780microns.

In general, the bone, such as, for example, cortical bone, is harvestedfrom any bone bearing source. Such sources include, but are not limitedto, the iliac crest, femur, humerus, tibia, fibula, radius, ulna, andribs. The bone, such as, for example, cortical bone, is removed from itssource, and then is cut or milled into particles. The particles then maybe separated according to size, such as by passing the particles throughone or more sieves. For example, the particles may be passed through a1,500 micron sieve, then through a 780 micron sieve, and then through a125 micron sieve. Such sieving provides a portion of bone particleshaving a D₉₀ from 780 microns to 1,500 microns, and another portion ofbone particles having a D₉₀ from 125 microns to 780 microns.

The sieved bone then is treated with at least one demineralization agentfor a time and at a concentration to expose osteogenic proteins presentin the cortical bone matrix. In one embodiment, the bone particles, suchas, for example, cortical bone particles, are treated with hydrochloricacid at a concentration of about 0.5N for a period of time of from about15 minutes to 180 minutes. In one embodiment, the bone particles, suchas, for example, cortical bone particles, are treated with hydrochloricacid at a concentration of about 0.5N for a period of time of from about30 minutes to 120 minutes. In one embodiment, the bone particles, suchas, for example, cortical bone particles, are treated with hydrochloricacid at a concentration of about 0.5N for a period of time of from about50 minutes to 70 minutes.

In one embodiment, subsequent to the treatment with the at least onedemineralization agent, the bone, such as, for example, cortical bone istreated with one or more antibiotics or one or more antimycotics inorder to reduce the level of bioburden within the bone. Antibiotics andantimycotics which may be employed include, but are not limited to,those mentioned hereinabove with respect to treatment of the cancellousbone.

After the bone, such as, for example, cortical bone, has been treated ashereinabove described, it then is combined with the cancellous bone toprovide a bone implant product.

In one embodiment, the bone implant includes at least about 50 vol. % ofthe cancellous bone hereinabove described, and up to about 50 vol. % ofthe demineralized bone, such as demineralized cortical bone, ashereinabove described. In another embodiment, the bone implant includesfrom about 55 vol. % to about 85 vol. % of the cancellous bone, and fromabout 15 vol. % to about 45 vol. % of the demineralized bone. In yetanother embodiment, the bone implant includes from about 55 vol. % toabout 65 vol. % of the cancellous bone, and from about 35 vol. % toabout 45 vol. % of the demineralized bone.

In one embodiment, the bone implant includes at least about 50 vol. % ofthe cancellous bone hereinabove described, from about 5 vol. % to about40 vol. % of demineralized bone particles, such as demineralizedcortical bone particles, having a D₉₀ from about 780 microns to about1,500 microns, and from about 5 vol. % to about 20 vol. % ofdemineralized bone particles, such as demineralized cortical boneparticles, having a D₉₀ from about 125 microns to about 780 microns.

In one embodiment, the cancellous bone is present in the bone implant inan amount of from about 55 vol. % to about 85 vol. %. In anotherembodiment, the cancellous bone is present in the bone implant in anamount of from about 55 vol. % to about 65 vol. %. In still anotherembodiment the cancellous bone is present in the bone implant in anamount of from about 57 vol. % to about 65 vol. %.

In one embodiment, the demineralized bone particles, such asdemineralized cortical bone particles, having a D₉₀ from about 780microns to about 1,500 microns are present in the bone implant in anamount of from about 5 vol. % to about 30 vol. %. In another embodiment,the demineralized bone particles having a D₉₀ from about 780 microns toabout 1,500 microns are present in the bone implant in an amount of fromabout 24 vol. % to about 28 vol. %.

In one embodiment, the demineralized bone particles, such asdemineralized cortical bone particles, having a D₉₀ from about 125microns to about 780 microns are present in the bone implant in anamount of from about 8 vol. % to about 18 vol. %. In yet anotherembodiment, the demineralized bone particles having a D₉₀ from about 125microns to about 780 microns are present in the bone implant in anamount of about 18 vol. %.

The bone implant including the cancellous bone and demineralized bone,such as, for example, demineralized cortical bone, may be added to anappropriate preservation medium, such as a cryopreservation medium orvitrification medium as hereinabove described, in which the bone implantmay be stored prior to use.

Such bone implant, which includes the cancellous bone and demineralizedbone as hereinabove described, may be used to treat the bone defects,disorders, and injuries hereinabove described, and may be administereddirectly to the site of the bone disease disorder, defect, or injury ashereinabove described to treat such disease, disorder, defect, orinjury.

The invention now will be described with respect to the followingexamples. It is to be understood, however, that the scope of the presentinvention is not intended to be limited thereby.

EXAMPLE 1

A bone sample was removed from a saline holding solution afterdebridement of soft tissue, and swabbed with a wiper. The bone wasplaced in a cutting area, and positioned face up. The bone then was cutwith a reciprocating saw to remove the cortical shell. The remainingbone then was placed into a 500 ml washing bag filled halfway withsaline (0.9%) and anticoagulant citrate dextrose solution, Formula A(ACD-A), at a ratio of 9 parts 0.9% saline to one part ACD-A. The baglid then was closed, and shaken vigorously. The bone sample then wasretrieved from the washing solution, and then placed into a bone millingchamber. The bone was then milled, and then the milled bone was spoonedout, and placed into a 500 ml receiver bottle containing up to 300 ml of0.9% saline and ACD-A at a volume ratio of 9 parts saline to one partACD-A.

The milled bone then was washed. First, the wash solution was decantedfrom the receiving bottle into a waste beaker. The wash solution wasreplaced with a new wash solution of 0.9% saline and ACD-A (volume ratioof 9 parts saline to one part ACD-A), the lid on the receiver bottle wastightened, and the bottle was shaken vigorously. The above washing wasrepeated until the wash solution was clear, and the chips of milled bonewere white to off-white in color. The last wash solution was decanted,and the bone chips were removed from the receiver bottle with a samplerspoon. Using a Petri dish and forceps, bone samples then were separatedout from large blood clot pieces and tissues. The bone then was packedinto a 50 ml conical tube, and the total volume was recorded. The totalvolume of bone chips was split by separating half for collagenasetreatment and half for fresh chips. The chips designated for collagenasetreatment were placed into a sterile 500 ml receiver bottle, and thefresh chips were placed into a 500 ml receiver bottle and covered with0.9% saline and ACD-A at a volume ratio of 9 parts saline to one partACD-A.

A collagenase solution then was prepared by mixing a collagenase powderwith 1× Phosphate Buffered Saline (“PBS”) in an amount of 1 mgcollagenase for each milliliter of PBS. The collagenase solution wasadded to the milled bone designated for the collagenase treatment in anamount of 2 ml collagenase solution for each cc of bone to be treated.The bottle containing the milled bone and the collagenase solution thenwas placed, with the lid loose, onto a rocker inside a 37° C. incubatorfor 10 minutes±1 minute. The bottle then was removed from the incubator,and the collagenase solution was decanted into a bottles. Thecollagenase treated milled bone then was rinsed by adding the samevolume of PBS and ACD-A at a volume ratio of 9 parts PBS to one partACD-A. This rinsing solution then was decanted into the bottlecontaining the collagenase solution. The rinsing then was repeated, therinsing solution again was decanted, and the collagenased bone samplewas added to the bottle of fresh bone sample. The resulting combined(collagenase treated plus fresh) bone sample then was poured through a 1mm sieve and placed in a clean bottle. The combined bone sample then wasrinsed with Dulbecco's Minimal Essential Medium (DMEM), low glucose,with phenol, prior to antibiotic treatment. A demineralized bone samplealso was rinsed with DMEM prior to antibiotic treatment.

A 1× antibiotic solution then was prepared. For each 1 ml of solution,0.9 ml of DMEM was mixed with 0.005 ml of 10 mg/ml gentamicin sulfate,0.05 ml of 50 mg/ml vancomycin HCl, and 0.01 ml of 250 μg/mlamphotericin B. Thus, the antibiotic solution included gentamicinsulfate at a concentration of 50 μg/ml, vancomycin HCl at aconcentration of 50 μg/ml, and amphotericin B at a concentration of 2.5μg/ml.

The antibiotic solution then was added to each of the combined bonesample and the demineralized bone sample in an amount of 2 ml for each 1cc of bone sample. The bottles then were placed onto a rocker inside ofa 37° C. incubator, with the lids loose, for no less than 18 hours. Thebottles then were removed from the incubator, placed under a biologicalsafety container, and the antibiotic solutions were decanted from eachof the combined bone sample, and the demineralized bone sample, and eachof the bone samples then was washed with the same volume of PBS. Thesamples then were shaken vigorously. The rinse solutions from the PBSwash were retained, and the pH of each solution was determined. Thesamples were washed with PBS until the pH of the rinse solutions was inthe range of 5.0 to 7.5. After the PBS washing was completed, PBS wasdecanted from each of the bone samples, and the same volume of PlasmaLyte-A (Baxter) solution, which includes 140 meq/l Na⁺, 5 meq/l K+, 98meg Cl−, 3 meq Mg²⁺, and 27 meg/l acetate, was added to each bonesample. The bone samples were shaken vigorously, and the Plasma Lyte-Awashing was repeated twice for each sample.

Using a sampler spoon, a 50 ml conical tube was packed gently with thecombined (collagenased and fresh) bone sample, and a demineralized bonesample was packed gently into a 15 ml conical tube. 5 ml of acryopreservation solution was added to the 15 ml conical tube and shakenvigorously. Each ml of cryopreservation solution included 0.7 ml of 1×Plasma Lyte-A, 0.2 ml of 25% human serum albumin, and 0.1 ml of 1×dimethylsulfoxide (DMSO) (CryoServ.) The final concentration of humanserum albumin, therefore, was 5% and the final concentration ofdimethylsulfoxide was 10%. After shaking, the de mineralized bone samplewas transferred to the 50 ml conical tube holding the cancellous bone,and then 10 ml of cryopreservation solution were added to the 50 mlconical tube and shaken vigorously. The entire sample then was placed ina product dose jar. 5 ml of cryopreservation solution then were added tothe product dose jar. If the bone product were not covered withsolution, up to 5 ml more of cryopreservation solution were added. Thejar then was sealed. After the jar was sealed, the product dose jar wasplaced into a packaging bag. The bag then was sealed. The secondarypackaging bag and a package insert then were placed into a tertiarymailer package, which then was heat sealed. The mailer package then wasplaced into an −80° C. quarantine freezer until the bone product wasready to be used.

EXAMPLE 2

A bone product from a single donor was prepared as described in Example1, except that, instead of storing 50 ml samples at −80° C., fourteen 5cc doses were labeled and stored at −50° C.±5° C. and six 5 cc doseswere labeled and stored at −80° C.±5° C. Seventeen days after freezing,three of the doses stored at −50° C. and three of the doses stored at−80° C. were thawed.

The jars containing the frozen bone samples placed in a 37° C. waterbath until the entire frozen products were thawed. The jars were removedfrom the water bath immediately upon thawing, and sprayed with 70%isopropanol. The outsides of the jars then were dried. The jars thenwere transferred to a biological safety cabinet, the lids were removed,and the thawed cryopreservation solutions were aspirated directly fromthe jars. 25 ml of Dulbecco's Minimal Essential Medium-low glucose(DMEM-lg) then were added to each of the jars, and the containers wereswirled such that the DMEM-lg covered the entire bone samplescompletely. The DMEM-lg then was aspirated from each jar, and the entirebone samples were transferred to 50 ml conical tubes. An additional 25ml of DMEM-lg then were added to each of the conical tubes containingthe bone samples.

A 1 mg/ml collagenase solution (1 mg/1 ml PBS) was prepared in a 250 mlreceiver bottle, 2 ml of collagenase solution were prepared per 1 cc ofbone sample.

The DMEM-lg was aspirated from the 50 ml conical tubes containing thebone samples, and then 25 ml of PBS were added to each of the conicaltubes, and the tubes were swirled to wash away any remaining DMEM-lg.The washing with PBS was repeated as necessary to remove any DMEM-lgstill visible in the bone samples.

After the final PBS wash, the PBS was aspirated off and the bone sampleswere transferred to 250 ml receiver bottles containing the collagenasesolutions. The bottles then were placed onto a rocker inside a 37° C.incubator for 15 minutes±1 minute. The bottles then were removed fromthe incubator, and the collagenase solutions were pipetted over 70 μmcell strainers into 250 ml conical tubes. The collagenased bone samplesthen were rinsed with PBS at volumes equal to the original collagenasetreatments. The PBS rinses were pipetted over the 70 μm cell strainersinto the 250 ml conical tubes. The PBS rinsing was repeated twice, andthe conical tubes were placed in a centrifuge (Beckman #GS-6R) and spunat 1960 rpm for 8 minutes (at approximately 878 g). The conical tubesthen were transferred to the biological safety cabinet, and thesupernatants were aspirated. The pellets then were loosened gently bydragging the tubes across the tube racks. The pellets then wereresuspended in appropriate volumes (1 to 4 ml) of PBS/ACD-A (containing59 ml AC-A per 500 ml PBS).

Three of the samples that were frozen at −50° C. and three of thesamples that were frozen at −80° C. then were tested for cell viability.

Using a 2-20 μl Pipetman pipette (Gilson, No. P20), 20 μl aliquots ofthe resulting cell suspensions were added to a microcentrifuge tube. 20μl aliquots of 0.4% Trypan Blue then were added to each of themicrocentrifuge tubes, and the contents were mixed by finger tapping. 10μl of the cell suspensions then were transferred into each side ofhemacytometers (Hausser Scientific, Model No. Steri-Cult 200) with acover slip in place. The cells then were allowed to settle in thecounting chambers before starting counts of viable and non-viable cells.The cells were not exposed to the Trypan Blue for more than 10 minutes,to prevent viable cells from absorbing the dye.

With the 10× objective of the microscope, the grid lines on each of thechambers' center 1 mm×1 mm squares were focused. The slides werepositioned such that the central areas of the grids were seen. Theobjective of the microscope then was changed to the 20× objective. Themicroscope then was focused, and viable and non-viable cells werecounted. Viable and non-viable cells in two chambers for each cellsuspension were counted. Viable and non-viable cell counts then weredetermined, from which were calculated the concentration of viablecells, the concentration of all cells, and the % viability of the cells.

In order to be acceptable for in vivo administration, the samples musthave a viability of at least 70%, and have a cell density of at least1×10³ cells/cc.

The viability results for each of the samples are given in Table 1below.

TABLE 1 Storage temperature % Viability Cells/cc 50 ± 5° C. 78.6 8.80 ×10⁵ 50 ± 5° C. 77.8 7.00 × 10⁵ 50 ± 5° C. 79.3 5.84 × 10⁵ 80 ± S° C.76.1 S.60 × 10⁵ 80 ± 5° C. 77.0 S.36 × 10⁵ 80 ± S° C. 81.1 6.16 × 10⁵

The above data shows that acceptable viability data were obtained foreach sample tested, and that cancellous bone prepared in accordance withthe present invention can be stored at −80° C.±5° C. at least forseventeen days.

With respect to some applications, surgeons may need to store thecancellous bone at temperature higher than −80° C., such as −50° C. Theabove data shows that the cancellous bone may be stored at −50° C. andstill retain acceptable viability.

EXAMPLE 3

23 samples of collagenase-treated bone were prepared as described inExample 1, except that the bone samples were treated with collagenase atconcentrations from 0.167 mg/ml to 3 mg/ml as given in Table 2 below,and for a period of time from 5 min. to 24 hrs., also given in Table 2below, and the bone samples were not frozen. The volume of each bonesample and the total volume of collagenase used to treat each bonesample also are given in Table 2 below.

The fresh bone samples were tested for % viability and cell density asdescribed in Example 2. The results are given in Table 2 below.

TABLE 2 Volume of Volume of Carrier for Bone Collagenase Sample DonorCollagenase Conc. Duration (cc) (ml) Viability Cell Yield Cells/cc Bone1 020505 DMEM 0.5 mg/ml 24 hrs 18 50 91.0% 6.12E+06 3.40E+05 2 020805DMEM 0.5 mg/ml 24 hrs 12.5 30 83.5% 1.68E+05 1.34E+04 3 121404 DMEM 0.5mg/ml 3 hrs 5 30 84.0% 4.80E+05 9.60E+04 4 121404 DMEM 1.5 mg/ml 3 hrs10 30 81.0% 4.40E+06 4.40E+05 5 012105 DMEM 3 mg/ml 3 hrs 10 30 86.0%6.24E+06 6.24E+05 6 020805 DMEM 3 mg/ml 3 hrs 12.5 30 91.0% 1.62E+061.30E+05 7 011305 DMEM 3 mg/ml 3 hrs 10 30 86.0% 1.53E+05 1.53E+04 8020505 DMEM 3 mg/ml 3 hrs 10 50 77.6% 9.35E+06 9.35E+05 9 121804 DMEM0.167 mg/ml 3 hrs 20 60 74.0% 4.77E+05 2.39E+04 10 121804 DMEM 0.833mg/ml 3 hrs 20 60 78.0% 2.65E+06 1.33E+05 11 B-031505 DMEM 3 mg/ml 1 hr20 60 n/a 1.00E+06 5.00E+04 12 022405 DMEM 3 mg/ml 3 hrs 25 50 76.0%1.20E+05 4.80E+03 13 030905 DMEM 3 mg/ml 3 hrs 20 50 94.0% 1.40E+076.99E+05 14 022405 DMEM 3 mg/ml 3 hrs 10 25 94.7% 3.63E+05 3.63E+04 15030905 DMEM 3 mg/ml 24 hrs 10 30 92.8% 3.59E+06 3.59E+05 16 040705Saline 3 mg/ml 15 min 10 30 66.0% 1.30E+05 1.30E+04 17 040705 PBS 3mg/ml 10 min 5 30 90.4% 2.08E+05 4.16E+04 18 040705 PBS 3 mg/ml 20 min10 25 83.0% 1.83E+06 1.83E+05 19 040705 PBS 3 mg/ml 30 min 10 30 95.0%3.58E+06 3.58E+05 20 041305 PBS 3 mg/ml 5 min 15 30 92.1% 2.05E+061.37E+05 21 041305 PBS 3 mg/ml 10 min 15 30 96.2% 1.64E+06 1.09E+05 22041305 PBS 3 mg/ml 30 min 15 30 91.9% 3.83E+06 2.55E+05 23 041305 PBS 3mg/ml 1 hr 15 30 90.9% 1.78E+07 1.18E+06

The above data shows that cancellous bone treated with collagenase atvarious concentrations and for various periods of time in accordancewith the present invention retain acceptable viability.

EXAMPLE 4

In this example, two samples of cells obtained from cancellous bonesamples treated with collagenase are tested for their ability todifferentiate into osteogenic cells.

A stock of a standard culture medium was prepared by pipetting 111 ml ofFBS and 11 ml of antibiotic-antimycotic (Invitrogen Cat. No. 15240-062)into 1,000 ml of DMEM-lg to provide a medium having a finalconcentration of 10% FBS and 1% antibiotic-antimycotic.

A stock of a culture medium including osteogenic supplements (OS medium)was prepared by mixing 246 ml of the standard medium with 25 μl of 1 mMdexamethasone solution, 2.5 ml of 1M β-glycerophosphate (βGP) solution,and 1.25 ml of 10 mM ascorbic acid-2-phosphate (ASAP) solution in a 500ml sterile bottle. The materials are mixed by swirling the bottle gentlyfor 30 seconds. The medium then is poured into the reservoir of a 500 ml0.2μ filter with storage system. A vacuum line then is attached to the500 ml 0.2μ filter, and the medium is filter sterilized. The reservoirthen is removed and replaced with a cap. The OS medium is stored at 2°to 8° C.

A Fast Violet B solution was prepared by placing a Sigma® Fast Violet BSalt Capsule into 48 ml of distilled water. The resulting solution thenwas aliquoted into two 50 ml conical tubes with 12 ml of solution beingadded to each tube. The tubes then were stored at 4° C.

A citrate solution was prepared by diluting 2 ml of Sigma® CitrateConcentrated Solution to 100 ml with distilled water. The citratesolution was stored at 4° C.

Two collagenase-treated cancellous bone samples were prepared asdescribed in Example 1, and collagenase released (CR) cells from suchsamples were obtained as described in Example 2.

CR cells were plated at a density of 0.5-5.0×10⁶ cells per well in a6-well plate using 3 ml of the standard medium hereinabove described.The plate was labeled “PO”, and covered with a lid, and transferred to a37° C. incubator set at 5% CO₂ and 90%±5% humidity. Medium changes wereperformed twice weekly with a medium volume of 3 ml per well.

Starting on day 7, the plate was examined for cell growth. Confluentcultures were trypsinized for cells between day 14 and day 21.

An appropriate volume of trypsin was warmed to 20° C.-37° C. in a waterbath. The plate that is to be trypsinized was removed from theincubator. The medium was aspirated off, and 1.5 ml of trypsin was addedto each well to be trypsinized. The plate was placed back in the 37° C.incubator for 5 minutes. The plate then was removed from the incubator,and the ides and bottom of the plate were tapped to dislodge attachedcells. 3 ml of standard medium then was added to the wells, the cellsuspensions then were mixed, and the entire volumes from the wells weretransferred into a 5.0 ml conical tube. The wells then were rinsed with3 ml of standard medium, and the rinse medium was added to the tube. Thetotal volume of the tube then was brought to 45 ml.

The tube then was placed in a centrifuge, and spun at 1,960 rpm for 8minutes (about 878 g). The supernatant then was aspirated off, and thepellet was loosened gently by dragging the tube across a tube rack. Thepellet then was re-suspended in an appropriate volume of standard medium(about 1 to about 3 ml) for cell counting.

A 20 μl aliquot of the cell suspension then was tested for % viabilityand cell density as described in Example 2.

The cells then were plated at a density of 30×10³ cells (±15×10³ cells)per well in a 6-well plate using 3 ml standard medium. The plate thenwas labeled “OS”, covered with a lid, and transferred to a 37° C.incubator set at 5% CO₂ and 90%±5% humidity. The plate was incubated for24 to 48 hours.

The OS medium was warmed to 20°-37° C. in a water bath. The standardmedium then was aspirated off from each well of the plate, and 3 ml ofOS medium then was added to each well. The OS medium was changed everythird or fourth day, and the cultures were assayed for alkalinephosphatase expression between days 7 and 14.

Alkaline phosphatase staining then was conducted. Each well was rinsedtwice with 1 ml of PBS. A fixative solution then was prepared by mixingtwo volumes of citrate working solution with three volumes of acetone.Each well then was fixed for one minute with 1 ml of the fixativesolution.

0.5 ml of Sigma® alkaline solution naphthol AS-MX phosphate was added to12 ml of Fast Violet B solution. The solution then was covered withaluminum foil to protect it from light. 1 ml of the Fast VioletB/naphthol solution then was added to each well.

The plate then was incubated at room temperature, in the dark for onehour. The wells then were aspirated, and rinsed twice with 1 ml ofdistilled water. Cell cultures that exhibited a distinct pink stain insome or all of the cells were positive. Cell cultures from both samplesof cells gave positive results.

EXAMPLE 5

Cancellous bone, which included cancellous bone treated with collagenaseand cancellous bone that was untreated, was prepared as described inExample 1. The cortical bone was processed, milled, and washed accordingto normal standard operating procedure for tissue products. The corticalbone then was sieved through a 1,500μ sieve stacked atop a 780μ sievestacked atop a 125μ sieve stacked onto a receiving pan with WFI (waterfor injection) quality water, Larger fragments, herein referred to ascortical bone chips, which were above the 780μ sieve and below the1,500μ sieve were transferred to 50 mL conical tubes. Smaller fragments,herein referred to as cortical bone powder, which were above the 125μsieve and below the 780μ sieve, were transferred to another set of 50 mlconical tubes. After sieving, the cortical bone samples were processedseparately using the same methods described below. The samples arereferred to hereinbelow as cortical bone samples.

Cortical bone samples were treated with 0.5M hydrochloric acid for 70minutes, at a ratio of 1 cc of cortical bone fragments to 17 cc of 0.5Mhydrochloric acid (HCl) at 4° C. in roller bottles under constantrolling. The HCl was decanted off each sample and the samples wereneutralized with a 5% Sodium Phosphate/PBS solution. The sample wasshaken vigorously, and the 5% Sodium Phosphate/PBS solution was decantedoff. The sample was then rinsed with 1×PBS, shaken vigorously, and thepH of the PBS was taken. If the pH of the PBS were outside a 5-7.5 pHrange, the sample was rewashed with 1×PBS until the pH was between 5 and7.5 pH range, the sample was rewashed with 1×PBS until the pH wasbetween 5 and 7.5 pH. The bone sample then was rinsed through a 125 μMsieve and placed into a clean bottle. The sample was then treated with a1× antibiotic solution consisting of 0.9 ml of DMEM mixed with 0.005 mlof 10 mg/ml gentamicin sulfate, 0.05 ml of 50 mg/ml vancomycin HCl, and0.1 ml of 250 μg/ml amphotericin B resulting in a final antibioticsolution of gentamycin sulfate (50 μg/ml), vancomycin HCl (50 μg/ml) andamphotericin B (2.5 μg/ml). The bottles then were capped with a gaspermeable lid and transferred into a 37° C. incubator for 18+ hours. Thebottles then were removed from the incubator, placed into a BSC(biological safety cabinet), and the antibiotic solution was decantedoff. The bone samples were then washed with the same volume of PBS,shaken vigorously, and the PBS decanted off. The bone samples then werewashed three times in the same fashion with Plasmalyte-A. After washing,the bone samples were transferred to 50 ml conical tubes using samplerspoons.

Using a sampler spoon and tweezers, 7 cc cancellous bone, 3 cc ofcortical chips, and 2 cc of cortical powder processed as described abovewere added to a product dose jar. Cryopreservation solution, consistingof 1× Plasmalyte-A, 0.2 mL of 25% human serum albumin, and 0.1 ml of 1×dimethylsulfoxide (DMSO) (CryoServ) per m L, then was added to cover thematerial within the jar. The jar then was sealed tightly and shakenvigorously. After the jar was sealed, the product dose jar was placedinto a packaging bag and the bag was then sealed. The secondarypackaging bag then was placed into a storage containing and placed intoan −80° C. quarantine freezer until the product was used for furtherstudy.

EXAMPLE 6

Five lots of “test” bone samples, prepared as described in Example 5,and five lots of “control” bone samples, prepared as described inExample 1, were obtained from five donors. From each donor, a test bonesample and a control bone sample was prepared.

Prior to implantation, the bone samples were thawed either at roomtemperature, or in a water bath at a temperature no greater than 39° C.Once the bone samples were thawed completely, as much cryoprotectant wasdecanted off and discarded. Sterile saline then was added to the jars tocover the bone samples. The bone samples were implanted within 4 hoursof being thawed. The samples then were removed from the sterile saline,weighed, and were placed gently in sterile syringes.

25 athymic male nude rats, each weighing at least 100 grams at thebeginning of the study, were divided into five groups of 5 rats each.Each rat of each group received a test bone sample and a control bonesample from the same donor.

Each rat was given one pre-operative injection of buprenorphine (0.1mg/kg IP), and then anesthetized with inhalant isoflurane gas. The upperportion of the back then was clipped and scrubbed for surgery.Hemostasis was employed as necessary. Doses of anesthesia were given asneeded to maintain the proper plane of unconsciousness.

Incisions of approximately 1 cm were made in the skin over each scapula.Pockets were made in the subcutaneous tissue by blunt and sharpdissection. Approximately 300±25 mg of either the test bone sample orthe control bone sample were placed into each pocket. The subcutaneoustissue and skin then were dosed with sutures, After the rats recoveredfrom the anesthesia, they were returned to their cages. The ratsreceived one post-operative injection of buprenorphine (0.1 mg/kg IP)for analgesia on the day after surgery.

The rats were weighed and then euthanized on Day 28 with CO₂. Theimplant sites then were opened and observed in order to confirm theretention of the implanted bone samples. The bone samples with adjacentsoft tissue were excised, fixed, and processed for pathology.

Gross necropsy findings, and most critically, the microscopic analyseswere the main study endpoints determining the presence or absence ofosteoinduction at the implant sites.

Evaluation of osteoinduction was based on the successful implantation ofall bone samples and the survival of sufficient animals until the studyendpoint. Also, at recovery of the implants, the implant sites mustgrossly be free from bacterial contamination as evidenced by the absenceof exudates, inflammation, or frank tissue destruction (necrosis). Sitesthat were associated with local infection that resolved during thecourse of the study were considered suspect. Microscopic evidence of thebone sample must be present at the implant site.

The bone samples and the surrounding tissue were fixed in 10% neutralbuffered formalin (NBF) prior to routine histology, decalcification, andprocessing. The samples then were processed into paraffin, and sectionsfrom 3 to 6 microns thick were cut and mounted onto glass slides andstained with hematoxylin and eosin. Blocks were faced as necessary.Sections were taken from at least three levels within the block. Theslides were evaluated for evidence of osteoinduction. More particularly,the slides were evaluated for the presence of the following:

-   -   1. chondroblasts;    -   2. chondroytes;    -   3. osteoblasts;    -   4. osteocytes;    -   5. cartilage;    -   6. osteoid tissue;    -   7. new bone; and,    -   8. new bone marrow.

Evidence of osteoinduction was graded as follows:

-   -   0—no evidence of new bone formation    -   1—1-25% of field shows evidence of new bone formation    -   2—26-50% of field shows evidence of new bone formation    -   3—51-75% of field shows evidence of new bone formation    -   4—76-100% of field shows evidence of new bone formation

Samples having a score of 0 were considered non-osteoinductive whilesamples having a score of from 1 to 4 were considered osteoinductive.

The results for the test and control samples from each donor for eachrat are given in Table 3 below. As shown in Table 3, an “X” indicatesthe presence of the element, while a minus sign “−”, indicates that theelement was not present.

TABLE 3 Site Rat L OR CHONDROBLASTS/ OSTEOBLASTS/ CARTILAGE/ NEW BONEORIGINAL GRADE Number R CYTES CYTES OSTEOID BONE MARROW DBM (0-4) TESTARTICLE- DONOR 1 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R — X — X X X1 2 — — — — — X 0 3 — — — — — X 0 4 — — — — — X 0 5 — X — X — X 1 TESTARTICLE- DONOR 2 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R — X — X — X1 2 X X X X X X 1 3 X X X X X X 1 4 X X X X — X 1 5 — X — X X X 1 TESTARTICLE- DONOR 3 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R — — — — — X0 2 — — — — —  X* 0 3 X X X X X X 4 4 X X X X X X 1 5 — — — — — X 0 TESTARTICLE- DONOR 4 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R X X — X — X1 2 — — — — — X 0 3 X — X — — X 1 4 — — — — — X 0 5 X X X X X X 1 TESTARTICLE- DONOR 5 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 R — — — — — X0 2 X X X X X X 1 3 — X — X — X 1 4 X X X X X X 1 5 — — — — — X 0CONTROL ARTICLE- DONOR 1 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 L — —— — — X 0 2 — — — — — X 0 3 — — — — — X 0 4 — — — — — X 0 5 — — — — — X0 CONTROL ARTICLE- DONOR 2 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 L —X — X — X 1 2 — X — X X X 1 3 X X — X X X 1 4 — X X X X X 1 5 — — — — —X 0 CONTROL ARTICLE- DONOR 3 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 L— — — — — X 0 2 — — — — — X 0 3 — — — — — X 0 4 — — — — — X 0 5 — — — —X 0 CONTROL ARTICLE- DONOR 4 OBSERVED ELEMENTS OF NEW BONE FORMATION 1 L— X — X X X 1 2 — — — — — X 0 3 — X — X X X 1 4 — — — — — X 0 5 — — — —— X 0 CONTROL ARTICLE- DONOR 5 OBSERVED ELEMENTS OF NEW BONE FORMATION 1L — X — X — X 1 2 — — — — — X 0 3 — — — — — X 0 4 — — — — — X 0 5 — — —— — X 0

The results for each of the test and control articles are summarized asfollows:

Test Article from Donor 1—Evidence of osteoinduction, includingosteoblasts/osteocytes, and new bone, was found in two of the implants.Bone marrow also was found in one of the implants. Thus, the testarticle from Donor 1 was considered to be osteoinductive.

Test Article from Donor 2—Evidence of osteoinduction, includingosteoblasts/osteocytes and new bone, was found in all five of theimplants. Chondroblasts/chondrocytes, cartilage/osteoid tissue, and bonemarrow were found in three of the implants. Thus, the test article fromDonor 2 was considered to be osteoinductive.

Test Article from Donor 3—Evidence of osteoinduction, includingchondroblasts/chondrocytes, osteoblasts/osteocytes, cartilage/osteoidtissue, new bone, and bone marrow, was found in two of the implants.Thus, the test article from Donor 3 was considered to be osteoinductive.

Test Article from Donor 4—Evidence of osteoinduction, includingchondroblasts/chondrocytes, was found in three of the implants.Osteoblasts/osteocytes, cartilage/osteoid tissue, and new bone werefound in two of the implants. Bone marrow was found in one of theimplants. Thus, the test article from Donor 4 was considered to beosteoinductive.

Test Article from Donor 5—Evidence of osteoinduction, includingosteoblasts/osteocytes, was found in three of the implants.Chondroblasts/chondrocytes, cartilage/osteoid tissue, and bone marrowwere found in two of the implants. Thus, the test article from Donor 5was considered to be osteoinductive.

With respect to the control articles, no evidence of osteoinduction wasfound in any of the implant sites from the control articles from Donor 1and Donor 3. Thus, the control articles from Donor 1 and Donor 3 wereconsidered to be non-osteoinductive.

The results with respect to the remaining control articles were asfollows:

Control Article from Donor 2—Evidence of osteoinduction, includingosteoblasts/osteocytes and new bone, was found in four of the implants.Bone marrow was found in three of the implants, andchondroblasts/chondrocytes and cartilage/osteoid tissue were found inone of the implant sites. Thus, the control article from Donor 2 wasconsidered to be osteoinductive.

Control Article from Donor 4—Evidence of osteoinduction, includingosteoblasts/osteocytes, new bone, and bone marrow, was found in two ofthe implant sites. Thus, the control article from Donor 4 was consideredto be osteoinductive.

Control Article from Donor 5—Evidence of osteoinduction, includingosteoblasts/osteocytes and new bone, was found in one of the implantsites. Thus, the control article from Donor 5 was considered to beosteoinductive.

EXAMPLE 7

The test and control samples from Donors 1 through 5 as described inExample 6 hereinabove were tested for cell density and cell viability asdescribed in Example 2 hereinabove. The cell density results are givenin Table 4 below, and the cell viability results are given in Table 5below.

TABLE 4 Cell Density (cells/cc) Donor Control Sample Test SampleDifference 1 7.07 × 10⁶ 4.38 × 10⁶ −2.69 × 10⁶ 2 1.43 × 10⁶ 2.48 × 10⁶ 1.05 × 10⁶ 3 4.07 × 10⁶ 3.16 × 10⁶ −0.93 × 10⁶ 4 2.47 × 10⁶ 2.24 × 10⁶−0.23 × 10⁶ 5 1.23 × 10⁶ 4.05 × 10⁶  2.82 × 10⁶

TABLE 5 Cell Viability Donor Control Sample Test Sample Difference 190.2% 82.0% −8.2% 2 80.6% 68.6% −12.0% 3 86.1% 80.5% −5.6% 4 88.3% 79.6%−8.7% 5 82.4% 79.9% −2.5%

EXAMPLE 8

The control and test samples from each of Donors 1 through 5 were testedfor osteodifferentiation in a revision of the method described inExample 4 hereinabove. In this example, two bone samples from each ofthe control and test samples from Donors 1 through 5 of cells wereobtained from cancellous bone samples treated with collagenase aretested for their ability to differentiate into osteogenic cells.

A stock of a standard culture medium was prepared by pipetting 111 ml ofFBS and 11 ml of antibiotic-antimycotic (Invitrogen Cat. No. 15240-062)into 1,000 ml of DMEM-low glucose (DMEM-lg) to provide a medium having afinal concentration of 10% FBS and 1% antibiotic-antimycotic.

A stock of a culture medium including osteogenic supplements (OS medium)was prepared by mixing 246 ml of the standard medium with 25 μl of 1 mMdexamethasone solution, 2.5 ml of 1M β-glycerophosphate (βGP) solution,and 1.25 ml of 10 mM ascorbic acid-2-phosphate (AsAP) solution in a 500ml sterile bottle. The materials are mixed by swirling the bottle gentlyfor 30 seconds. The medium then is poured into the reservoir of a 500 ml0.2μ filter with storage system. A vacuum line then is attached to the500 ml 0.2μ filter, and the medium is filter sterilized. The reservoirthen is removed and replaced with a cap. The OS medium is stored at2°-8° C.

A BCIP/NBT working solution was prepared by placing 1 drop each ofReagent 1, 2, and 3 from the BCIP/NBT Alkaline Phosphatase Substrate KitIV into 5 mL of Tris-HCL Buffer 100 mM. The resulting solution then wasaliquoted into a 50 ml conical tube and stored at 4° C.

One of the two collagenase-treated cancellous bone samples (BoneSample 1) was prepared as described in Example 1, and collagenasereleased (CR) cells from such samples were obtained as described inExample 2. The cryopreservation solution from Bone Sample 1 was placedinto a conical tube and set aside. The other bone sample (Bone Sample 2)was thawed in a water bath at 37° C.) and then the cryopreservationsolution was aspirated off and added to the conical tube containing thecryopreservation solution from Bone Sample 1. 10 mL of standard mediumwas then added to the Bone Sample 2 container, the sample was shaken,and the medium was transferred to the conical tube containing thecryopreservation solution from Bone Sample 1 and 2. Then the tube wasplaced in a centrifuge, and spun at 1,960 rpm for 8 minutes (about 878g). The media was then aspirated from the tube and the cells wereresuspended in 4 mL of standard medium and placed into a well in a 6well plate labeled ‘Cells from Cryopreservation Solution (Samples 1 &2). The remaining bone chips of Bone Sample 2 were then transferred toanother well in the 6 well plate labeled Osteogenic Sample 2 Bone Chips.

CR cells were plated into one well of the six well plate in 4 mL ofstandard medium hereinabove described. The well was labeled as CR CellsSample 1. Bone samples were placed into 3 wells of the same 6 well plateat 1.5-2 cc per well with 4 mL of standard media per well and all threewells were labeled as Osteogenic Sample 1. The plate was covered with alid, and transferred to a 37° C. incubator set at 5% CO₂ and 90%±5%humidity. Medium changes were performed every 2-4 days with a mediumvolume of 4 ml per well.

Plates were examined daily until confluence was greater than 60%. Onceconfluence was greater than 60%, plates were trypsinized for cells.

An appropriate volume of trypsin was warmed to 37° C. in a water bath.The plate that is to be trypsinized was removed from the incubator. Themedium as aspirated off, and each well containing bone chips was washedwith 3.5 mL of PBS and the well containing cells with 3 mL. The PBS wasthen aspirated and 2 mL of trypsin was added to each well containingbone chips and 1.5 mL to each well containing cells. The plate wasplaced back in the 37° C. incubator for 5 minutes. The plate then wasremoved from the incubator, and the sides and bottom of the plate weretapped to dislodge attached cells. 3 ml of standard medium then wereadded to the wells, the cell suspensions then were mixed, and the entirevolumes from the wells were transferred into a 50 ml conical tube. Thewells then were rinsed with 3 ml of standard medium, and the rinsemedium was added to the tube. The total volume of the tube then wasbrought to 45 mL The tube then was placed in a centrifuge, and spun at1,960 rpm for 8 minutes (about 878 g). The supernatant then wasaspirated off, and the pellet was loosened gently by dragging the tubeacross a tube rack. The pellet then was re-suspended in 9 mL of standardmedium. 3 mL of the cell suspension was plated in each of 3 wells of a 6well plate and the plate was covered and transferred to a 370 incubator.The plate then was labeled “OS”, covered with a lid, and transferred toa 37° C. incubator for 24 to 48 hours.

The OS medium was warmed to 37° C. in a water bath. The standard mediumthen was aspirated off from each well of the plate, and 3 ml of OSmedium then was added to each well. The OS medium was changed everythird or fourth day, and the cultures were assayed for alkalinephosphatase expression between days 7 and 14.

Alkaline phosphatase staining then was conducted. Each well was rinsedtwice with 1 ml of PBS. 1 ml of BCIP/NBT Working solution was added toeach well and plates were incubated at room temperature in the dark for6-30 hours. All wells were then evaluated for the presence of theblue/purple color of the positive Alkaine Phosphatase reaction. Each ofthe test and control samples from each of Donors 1 through 5 waspositive for osteodifferentiation.

EXAMPLE 9

Evaluation of cortical chip size was performed through a series grindingand sifting procedures in which cortical bone was sorted into multiplesize ranges. The various sized cortical chip fragments were thencompared. Blinded evaluation of mixtures of cancellous chips andcortical chips of various chip sizes with varied cortical to cancellousratios was performed by seven people with previous experience inosteoimplantation techniques. Six of the seven persons evaluating thetrial mixtures selected the same chip size and formulation when asked toselect the sample having optimal physical characteristics, such optimalmixture was found to comprise 70% cancellous bone chips, and 30%cortical bone chips of 780 microns to 1500 microns in size, compared toa mixture of 100% cancellous bone chips, Blinded evaluation of thisoptimal mixture was then performed by orthopedic surgeons, operatingunder test conditions similar to typical surgical conditions, also foundproducts comprising this optimal mixture to exhibit superior handlingand physical characteristics.

The disclosures of all patents, publications (including published patentapplications), depository accession numbers, and database accessionnumbers were incorporated herein by reference to the same extent thateach patent, publication, depository accession number and databaseaccession number were specifically and individually incorporated byreference.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

What is claimed is:
 1. A bone implant, comprising: cancellous bone thatis essentially free of blood cells, wherein the cancellous bone has beentreated with a loosening agent configured to partially digest thecancellous bone, thereby loosening viable osteogenic cells from a bonematrix in the cancellous bone; and cortical bone that has been treatedwith a demineralization agent configured to expose osteoinductiveproteins of the cortical bone.
 2. The bone implant of claim 1, whereinthe demineralization agent comprises hydrochloric acid.
 3. The boneimplant of claim 1, wherein the loosening agent comprises collagenase.4. The bone implant of claim 1, wherein the cancellous bone is treatedwith the loosening agent by exposing the cancellous bone to theloosening agent for a period of time from about 5 minutes to about 3hours.
 5. A method of preparing a bone implant, the method comprising:providing a cancellous bone that is essentially free of blood cells,wherein the cancellous bone comprises viable osteogenic cells containedwithin a bone matrix; treating the cancellous bone with a looseningagent configured to partially digest the cancellous bone, therebyloosening the viable osteogenic cells from the bone matrix; providing acortical bone; treating the cortical bone with a demineralization agentconfigured to expose osteoinductive proteins of the cortical bone; andcombining the cancellous bone and the cortical bone in a container. 6.The method of claim 5, wherein the cortical bone comprises particleshaving a D₉₀ of less than about 1,500 microns.
 7. The method of claim 5,wherein the demineralization agent comprises hydrochloric acid.
 8. Themethod of claim 5, further comprising storing the cancellous bone, thecortical bone, and a cryopreservative in a sealed container.
 9. Themethod of claim 5, wherein the cancellous bone is treated with theloosening agent for a period of time from about 5 minutes to about 3hours.
 10. The method of claim 5, wherein the loosening agent comprisescollagenase having a concentration in a range from about 0.1 mg/ml toabout 3.0 mg/ml.
 11. The method of claim 5, wherein the loosening agentcomprises a digestive enzyme selected from the group consisting oftrypsin, amylase, and lipase.
 12. The method of claim 5, furthercomprising treating the cancellous bone with at least one antibioticcompound.
 13. The method of claim 12, wherein the at least oneantibiotic compound is selected from the group consisting of gentamicin,vancomycin, penicillin, erythromycin, and combinations thereof.
 14. Themethod of claim 5, further comprising treating the cancellous bone withat least one antimycotic compound.
 15. The method of claim 14, whereinthe at least one antimycotic compound is selected from the groupconsisting of amphotericin, fluconazole, and combinations thereof. 16.The method of claim 5, further comprising harvesting the cancellous bonefrom a source and removing a cortical shell of the cancellous bone. 17.The method of claim 5, wherein the cancellous bone is at least 50% byvolume of the bone implant.